1. Field of the Invention
This invention relates to multiphoton-excitation observation apparatuses.
This application is based on Japanese Patent Application No. 2006-088760, the content of which is incorporated herein by reference.
2. Description of Related Art
A multiphoton-excitation apparatus is a known apparatus in the related art for observing the function of cells and so forth by irradiating a biological specimen or the like with ultrashort pulsed laser light from the surface thereof and detecting the fluorescence emitted from the surface or the interior of the specimen due to multiphoton absorption (for example, see Publication of Japanese Patent No. 2848952).
Also known in the related art are scanning laser microscopes which acquire image data in a two-dimensional XY plane by detecting reflected light or transmitted light from a specimen while scanning a spot of light in the two-dimensional XY plane on the specimen, and which acquire image data at different depths in the specimen, ranging from a surface layer portion to a deep layer portion, by varying the focal position of the spot of light in the Z direction.
However, when the focal position is varied in the depth direction in this way, there is a phenomenon whereby the brightness decreases the deeper each acquired image becomes in the Z-direction. Conventionally, therefore, a technique for correcting the apparent brightness by subjecting the light level of the image data acquired in the observation to post-processing to obtain easily observed images with uniform brightness has been proposed (for example, see Japanese Unexamined Patent Application, Publication No. 2000-275541).
As biological research has progressed in recent years, there have been increasing demands for observing regions deeper inside biological specimens. A standard single-photon-excitation laser scanning microscope can acquire an optical internal tomogram of a specimen under observation by virtue of the confocal effect; however, the observable depth in the biological specimen is only about 50 μm from the surface.
A multiphoton excitation method described in Publication of Japanese Patent No. 2848952 excites fluorescence at near-infrared wavelengths, which have high transmittance through biological tissue and, therefore, are suitable for observing deep regions. However, even with multiphoton excitation, when observing a deep region exceeding a depth of 100 μm, for example, the effects of scattering, refraction, and absorption of the excitation light and the fluorescence by the specimen are strong, resulting in the problem that the resolution and S/N ratio decrease, which makes it impossible to acquire clear images.